Inhibiting GS-FDH to modulate no bioactivity

ABSTRACT

Patients needing NO donor therapy or inhibition of pathologically proliferating cells or increased NO bioactivity are treated with a therapeutically effective amount of an inhibitor of glutathione-dependent formaldehyde dehydrogenase.

RELATED APPLICATIONS

This patent application is a Continuation of U.S. application Ser. No.11/648,406, filed Dec. 29, 2006, which is a Divisional of U.S.application Ser. No. 09/757,610, filed Jan. 11, 2002, now issued U.S.Pat. No. 7,179,791, each of which are herein incorporated by referencein their entireties.

TECHNICAL FIELD

The invention relates to modulating NO (nitric oxide) bioactivity toobtain therapeutic effects.

BACKGROUND OF THE INVENTION

Stamler et al. U.S. Pat. No. 6,057,367 is directed to treating mammalsfor infections or for conditions associated with pathologicallyproliferating mammalian cell growth (for example, certain cancers,restenosis, benign prostatic hypertrophy) by administration of amanipulator of nitrosative stress (an impetus for NO or NO.sub.2 groupattachment to proteins, nucleic acids or other biological molecules) toselectively kill or reduce the growth of the microbes or helminthscausing the infection or of host cells infected with the microbes or ofthe pathologically proliferating mammalian cells.

Stamler et al. U.S. application Ser. No. 09/695,934 discloses use of NOdonors to prevent the occurrence of restenosis following angioplasty, toinhibit platelets to prevent coagulation and thrombus formation, totreat angina in patients at risk for coagulation and thrombus formation,to inhibit microbes, to treat impotence, asthma, cystic fibrosis,hypoxia and ischemic disorders, heart failure, stroke, arthritis, ARDS,hypertension, neurodegeneration, painful crisis of sickle cell disease,cancer and any pathological proliferation of cells and any NMDA relatedinjury. The invention is directed to C-nitroso compounds and use thereofas NO donors.

Gaston, Stamler and Griffith U.S. application Ser. No. 08/403,775 isdirected to use of inhibitors of S-nitrosothiol breakdown to treatasthma.

Numerous enzymes have been shown to break down S-nitrosothiols in vitro.These include (a) thioredoxin system (b) glutathione peroxidase, (c)gamma glutamyl transpeptidase, (d) xanthine oxidase, (e) alcoholdehydrogenase Class III, and (f) other classes of alcohol dehydrogenase.

In respect to alcohol dehydrogenases including alcohol dehydrogenaseClass III (also known as glutathione-dependent formaldehydedehydrogenase), see the following publications which present in vitrodata: Kuwada, M., et al. J. Biochem. 88, 859-869 (1980); Jensen, D. E.,et al., Biochemical Pharmacology 53, 1297-1306 (1997); Jensen, 1). E.,et al., Biochem. J. 331, 659-668 (1998).

Despite the in vitro results referred to above, the current perspectiveis that thiols, ascorbate and copper ions break down S-nitrosothiols invivo. However, there has been no work heretofore demonstrating howS-nitrosothiols are broken down in vivo.

It has not heretofore been known that inhibition ofglutathione-dependent formaldehyde dehydrogenase mediates NO donortherapy, nitrosative stress and NO bioactivity in vivo.

SUMMARY OF THE INVENTION

It has been concluded in the course of making the invention herein thatenzyme, namely glutathione-dependent formaldehyde dehydrogenase knownheretofore to oxidize the formaldehyde glutathione adduct,S-hydroxymethylglutathione, previously thought to be the major enzymesubstrate, fractions in vivo to metabolize S-nitrosoglutathione andprotein S-nitrosothiols to modulate NO bioactivity, by controlling theintracellular levels of low mass NO donor compounds and preventingprotein nitrosylation from reaching toxic levels.

Based on this, it follows that inhibition of the enzyme potentiatesbioactivity in all diseases in which NO donor therapy is indicated,inhibits the proliferation of pathologically proliferating cells, andincreases NO bioactivity in diseases where this is beneficial.

One embodiment herein is directed to a method of treating a patientafflicted with a disorder ameliorated by NO donor therapy, said methodcomprising administering to said patient a therapeutically effectiveamount of an inhibitor of glutathione-dependent formaldehydedehydrogenase.

Another embodiment herein is directed to a method for treating a patientafflicted with pathologically proliferating cells, said methodcomprising administering to said patient a therapeutically effectiveamount of an inhibitor of glutathione-dependent formaldehydedehydrogenase. The term “pathologically proliferating cells” is usedherein to include pathologic microbes, pathologic helminths, andpathologically proliferating mammalian cells.

Still another embodiment herein is directed to increasing NO bioactivityfor pharmacological effect or in the case of diseases associated with adeficiency of NO. This embodiment is directed to a method of treating apatient in need of increased nitric oxide bioactivity, said methodcomprising administering to said patient a therapeutically effectiveamount of an inhibitor of glutathione-dependent formaldehydedehydrogenase.

DETAILED DESCRIPTION

Glutathione-dependent formaldehyde dehydrogenase (GS-FDH) is a knownenzyme which is conserved from microbes including bacteria and fungi tomammals. It is also known as alcohol dehydrogenase Class III. It hasbeen identified in a variety of bacteria, yeasts, plants and animals.The proteins from E. coli, S. cerevisiae and mouse macrophages shareover 60% amino acid sequence identity. In methylotrophic microorganisms,GS-FDH is induced by methanol to prevent formaldehyde accumulation. Thephysiological significance of formaldehyde oxidation by GS-FDH is lessclear in other microorganisms and animals. In the course of making theinvention herein GS-FDH associated NADH-dependent S-nitrosoglutathionereductase activity has been detected in E. coli, in mouse macrophages,in mouse endothelial cells, in mouse smooth muscle cells, in yeasts, andin human HeLa, epithelial and monocyte cells. As used herein, the term“glutathione-dependent formaldehyde dehydrogenase” means enzyme thatoxidizes S-hydroxymethylglutathione and also provides NADH-dependentS-nitrosoglutathione reductase activity (i.e., decomposesS-nitrosoglutathione when NASH is present as a required cofactor) andshares at least 60% amino acid sequence identity with eves having thesame function from E. coli, S. cerevisiae and mouse macrophages. Theglutathione-dependent formaldehyde dehydrogenase may also be referred toas S-nitrosoglutathione reductase.

We turn now to the inhibitors of glutathione-dependent formaldehydedehydrogenase for use in the three embodiments herein. These compoundsinhibit the S-nitrosoglutathione reductase activity of theglutathione-dependent formaldehyde dehydrogenase.

One class of compounds for use herein as the inhibitors ofglutathione-dependent formaldehyde dehydrogenase is constituted ofcompetitors for NAD⁺ binding. These inhibitors work by binding to theNAD⁺ cofactor binding site of the enzyme and thereby block the NADHcofactor from binding to the enzyme.

One compound of this class is nicotinamide riboside (NR) which has thestructure:

Other compounds of this class include the following ribonucleosideanalogs:

The compound 6-aminonicotinamide (6AN) which has the structure:

This compound requires additionally metabolization to 6-amino-NAD(P⁺) bythe pentose phosphate pathway (PPP) enzyme, 6-phosphogluconatedehydrogenase, for inhibitory activity.

The compound 5-β-D-ribofuranosylnicotinamide which has the structure:

This compound requires conversion to the corresponding NAD analog,C-NAD, which is described later as Compound (13), for inhibitoryactivity.

The compound 6-β-D-ribofuranosylpicolinamide which has the structure;

This compound requires conversion to the corresponding NAD analog,C-NAD, which is described later, as Compound (14), for inhibitoryactivity.

The compound 2-(3-D-ribofuranosylisonicotinamide which has thestructure:

Other inhibitors of glutathione-dependent formaldehyde dehydrogenasewhich are ribonucleoside analogs and are competitors for NAD⁺ bindingand thereby inhibit the S-nitrosoglutathione reductase activity ofGS-FDH have the formula:

These include thiophenfurin(5-β-D-ribofuranosylthiophene-3-carboxamide), Compound (6a), which hasthe formula (6) where X=S and Y=CH; furanfurin(5-β-D-ribofuranosylfuran-3-carboxamide), Compound (6b), which has theformula (6) where X=O and Y=CH; tiazofurin(2-β-D-ribofuranosylthiazole-4-carboxamide), Compound (6c), which hasthe formula (6) where X=S and Y=N; selenazofurin(2-β-D-ribofuranosylselenazole-4-carboxamide), Compound (6d), which hasthe formula (6) where X=Se and Y=N; and selenophenfurin(5-β-D-ribofuranosylselenophene-3-carboxamide), Compound (6e), which hasthe formula (6) where X=Se and Y=CH. These compounds are metabolized totheir isosteric NAD analogs for activity. For example, tiazofurin isphosphorylated by adenosine kinase to the 5′-monophosphate and convertedby NAD-pyrophosphorylate to TAD, described later, for competitivebinding.

Still another ribonucleoside analog which is a competitor for NAD⁺binding and thereby inhibits the S-nitrosoglutathione reductase activityof GS-FDH is benzamide ribosome (BR) which has the formula.

(BR) is metabolized to (BAD), described later, for activity.

Still another ribonucleoside analog which is a competitor for NAD⁺binding and thereby inhibits the S-nitrosoglutathione reductase activityof GS-FDH is ribavirin which has the formula:

Still another ribonucleoside analog which is a competitor for NAD⁺binding and thereby inhibits the S-nitrosoglutathione reductase activityof GS-FDH is mizoribine (MZR) which has the formula.

Still another ribonucleoside analog for use herein as inhibitor ofGS-FDH is 5-ethynyl-1-β-D-ribofuranosylimidazole-4-carboxamide (EICAR)which has the formula:

This compound metabolizes to (EAD), Compound (17) described later, forinhibitory activity.

Another compound which is an inhibitor of GS-FDH by virtue of being acompetitor for NAD⁺ binding is NAD⁺ which has the formula:

Still other compounds which are inhibitors of GS-FDH by virtue of beingcompetitors for NAD⁺ binding and thereby inhibit theS-nitrosoglutathione reductase activity of GS-FDH are NAD⁺ derivatives.

One such NAD⁺ derivative is 6-amino-NAD which has the formula:

Another such NAD⁺ derivative is 5-β-D-ribofuranosylnicotinamide adeninedinucleotide (C-NAD) which has the formula:

This compound is a metabolite of Compound (3) described above.

Another such NAD⁺ derivative is 6-β-D-ribofuranosylpicolinamide adeninedinucleotide (C-PAD) which has the formula:

This compound is a metabolite of Compound (4) described above.

Other such NAD⁺ derivatives have the structural formula:

These include TFAD, Compound (15a), which has the formula (15) where Xis S and Y is CH and is a metabolite of Compound (6a); FFAD, Compound(15b), which has the formula (15) where X=O and Y=CH and is a metaboliteof Compound (6b); TAD (thiazole-4-carboxamide adenine dinucleotide),Compound (15c), which has the formula (15) where X=S and Y=N and is ametabolite of Compound (6c); SFAD, Compound (15d), which has the formula(15) where X=Se and Y=N, and is a metabolite of Compound (6d); and SAD,Compound (15e), which has the formula (15) where X=Se and Y=CK and is ametabolite of Compound (6e).

Yet another such NAD⁺ derivative is benzamide adenine dinucleotide (BAD)which has the formula:

Compound (16) is a metabolite of (BR) which is Compound (7) describedabove.

Yet another such NAD⁺ derivative is (EAD) which has the formula:

Compound (17) is a metabolite of EICAR, Compound (10) described above.

Still other such NAD⁺ derivatives have the formula:

These include β—CH₂-TAD, Compound (18a), which has the formula (18)where X and Y=OH, W=H and Z=CH₂; β-CF₂-TAD, Compound (18b), which hasthe formula (18) where X and Y=OH, W=H and Z=CF₂; 3′F-TAD, Compound(18c), which has the formula (18) where X=OH, Y=F, W=H and Z=O;2′Fara-TAD, Compound (18d), which has the formula (18) where X and Y=OH,W=F and Z=O; 2′Fara-β-CH₂-TAD, Compound (18e), which has the formula(18) where X and Y=OH, W=F and Z=CH₂; and 2′Fara-β-CF₂-TAD, Compound(18f), which has the formula (18) where X and Y=OH, W=F and Z=CF₂.

Still other such NAD⁺ derivatives have the formula:

These include β-CH₂—BAD, Compound (19a), which has the formula (19)where X=OH, W=H and Z=CH₂, and 2′Fara-β-CH₂—BAD, Compound (19b) whichhas the formula (19) where X=H, W=F and Z=CH.

The derivatives of formulas (18) and (19) including Compounds (18a),(18b), (18c), (18d), (18e), (18f), (19a) and (19b) are TAD (Compound(15c)) and BAD (Compound (16)) analogs which are metabolically stableand cell membrane permeable, methylene or difluoromethylenebis(phosphonate)s, and analogs substituted with fluorine in the ribosemoiety of adenosine which are more hydrophobic than their hydroxycongeners.

Other compounds which are inhibitors of GS-FDH are mimickers of thenicotinamide portion of NAD and a water molecule and includemycophenolic acid (MPA), Compound (20), and its morpholinoethyl esterprodrug mycophenolate mofetil (MMF), Compound (21).

Compound (20) has the formula:

Compound (21) has the formula:

Still other compounds which are inhibitors of GS-FDH are competitivesubstrates for NADH binding. These include 6-thioanologs of naturalpurine bases, e.g., 6-mercaptopurine (Compound 22) and 6-thioguanine(Compound 23).

Compound (22) has the formula:

Compound (23) has the formula:

The above Compounds (1)-(23) are considered also to inhibit the activityof other NADH dependent dehydrogenases such as inosine monophosphatedehydrogenase (IMPDH). Other inhibitors of IMPDH by virtue ofcompetition for NAD⁺ cofactor binding site, are also effective asinhibitors of GS-FDH herein.

The compounds specifically described above are available commercially ortheir synthesis is described in or obvious from the literature.

Some of the above compounds have been utilized for some of the utilitiesherein without knowledge that at least part of their function may havebeen due to GS-FDH inhibition; these compounds for these uses areexcluded from the invention herein, but are not excluded from theinvention herein for other uses.

For example, tiazofurin has been used previously for antineoplasticactivity against tumors as has thiophenfurin and selenazofurin.Moreover, selenazofurin, ribavirin, MZR, EICAR and MMF have been usedfor antiviral or potential antiviral activity. Moreover, mycophenolicacid has been evaluated as an anticancer, antiviral, antifungal andantibacterial agent, as well as for its therapeutic use in psoriasis andrheumatoid arthritis. Moreover, furanfurin and ribavirin have been shownto be inactive as an antitumor agents. These cases are excluded from theinvention herein. However, the same compounds are not excluded from theinvention herein for other uses.

Another class of compounds useful herein to inhibit GS-FDH isconstituted of glutathione derivatives including D-glutathione andS-alkyl glutathione containing from 1 to 6 carbon atoms in the S-alkylgroup.

The use of gold-based compounds to treat asthma and cystic fibrosis isexcluded from the invention herein.

We turn now to the embodiment directed to a method of treating a patientafflicted with a disorder ameliorated by NO donor therapy where themethod comprises administering to the patient a therapeuticallyeffective amount of an inhibitor of glutathione-dependent formaldehydedehydrogenase. This embodiment may be referred to as the firstembodiment herein.

The disorders applicable to this embodiment include, for example,breathing disorders (e.g., asthma, cystic fibrosis, and ARDS), heartdisease, hypertension, ischemic coronary syndromes, atherosclerosis,glaucoma, diseases characterized by angiogenesis (e.g., coronary arterydisease), disorders where there is risk of thrombosis occurring,disorders where there is risk of restenosis occurring, chronicinflammatory diseases (e.g., AID dementia and psoriasis), diseases wherethere is risk of apoptosis occurring (e.g., heart failure,atherosclerosis, degenerative neurologic disorders, arthritis and liverinjury (ischemic or alcoholic)), impotence, obesity caused by eating inresponse to craving for food, stroke, reperfusion injury (e.g.,traumatic muscle injury in heart or lung or crush injury), and disorderswhere preconditioning of heart or brain for NO protection againstsubsequent ischemic events is beneficial.

The inhibitors of glutathione-dependent formaldehyde dehydrogenase aredescribed above.

The term “therapeutically effective amount” for this first embodimentmeans a glutathione-dependent formaldehyde dehydrogenase inhibitingamount in vivo that causes amelioration of the disorder being treated orprotects against a risk associated with the disorder. For example, forasthma, a therapeutically effective amount is a bronchodilatingeffective amount; for cystic fibrosis, a therapeutically effectiveamount is an airway obstruction ameliorating effective amount; for ARDS,a therapeutically effective amount is a hypoxemia ameliorating effectiveamount; for heart disease, a therapeutically effective amount is anangina relieving or angiogenesis inducing effective amount; forhypertension, a therapeutically effective amount is a blood pressurereducing effective amount; for ischemic coronary disorders, atherapeutic amount is a blood flow increasing effective amount; foratherosclerosis, a therapeutically effective amount is an endothelialdysfunction reversing effective amount; for glaucoma, a therapeuticamount is an intraocular pressure reducing effective amount; fordiseases characterized by angiogenesis, a therapeutically effectiveamount is an angiogenesis inhibiting effective amount; for disorderswhere there is risk of thrombosis occurring, a therapeutically effectiveamount is a thrombosis preventing effective amount; for disorders wherethere is risk of restenosis occurring, a therapeutically effectiveamount is a restenosis inhibiting effective amount; for chronicinflammatory diseases, a therapeutically effective amount is aninflammation reducing effective amount; for disorders where there isrisk of apoptosis occurring, a therapeutically effective amount is anapoptosis preventing effective amount; for impotence, a therapeuticallyeffective is an erection attaining or sustaining effective amount; forobesity, a therapeutically effective amount is a satiety causingeffective amount; for stroke, a therapeutically effective amount is ablood flow increasing or a TIA protecting effective amount; forreperfusion injury, a therapeutically effective amount is a functionincreasing effective amount; and for preconditioning of heart and brain,a therapeutically effective amount is a cell protective effectiveamount, e.g., as measured by triponin or CPK.

In general, the dosage, i.e., the therapeutically effective amount,ranges from 1 μg to 10 g/kg and often ranges from 10 μg to 1 g/kg or 10μg to 100 mg/kg body weight of the patient, per day.

The patients include mammals including humans.

The preferred route of administration is oral administration althoughother routes of administration including parenteral are useful. Topicaladministration can be appropriate for localized disorders.

Preferred treating agents for the first embodiment includeD-glutathione, ribavirn, D-glutathione together with ribavirin, andmycophenolic acid. D-Glutathione can be given, for example,intravenously at 10-100 mg/kg and/or inhaled in 1-10 M concentration forasthma; inhaled at 1-10 mM concentration for cystic fibrosis and ARDS;and intravenously at 10-300 mg/kg for heart disease including angina,ischemic coronary syndrome, and disease where there is risk of apoptosisoccurring (e.g., acetaminophen induced liver injury), and at 100 to1,000 mg for hypertension. Ribavirin can be given, for example, injectedin an amount of 1-10 g at a concentration of 5-25 mg/ml for angina fromcoronary artery disease, inhaled in amount of 1 to 10 grams to preventthrombosis from occurring, e.g., where pulmonary embolism is found, andtopically at 1-5% in a topical composition for psoriasis. Mycophenolicacid can be given, for example, coated on a stent (drug concentration10-40% per polymer) for treating restenosis or topically at aconcentration of 1 to 10% in a paste to treat impotence.

Treatment is continued as long as symptoms and/or pathology ameliorate.

We turn now to the embodiment directed to a method of treating a patientafflicted with pathologically proliferating cells where the methodcomprises administering to said patient a therapeutically effectiveamount of an inhibitor of glutathione-dependent formaldehydedehydrogenase. This embodiment may be referred to as the secondembodiment herein.

We turn now to the case of the second embodiment herein where thepathologically proliferating cells are pathologically proliferatingmicrobes.

The microbes involved are those where glutathione-dependent formaldehydedehydrogenase is expressed to protect the microbe from nitrosativestress or where host cell infected with said microbe expresses saidenzyme thereby protecting the microbe from nitrosative stress.

The term “pathologically proliferating microbes” is used herein to meanpathologic microorganisms including but not limited to pathologicbacteria, pathologic viruses, pathologic Chlamydia, pathologic protozoa,pathologic Rickettsia, pathologic fungi, and pathologic mycoplasmata.More detail on the applicable microbes is set forth at columns 11 and 12of U.S. Pat. No. 6,057,367 which are incorporated here by reference.

The term “host cells infected with pathologic microbes” includes notonly mammalian cells infected with pathologic viruses but also mammaliancells containing intracellular bacteria or protozoa, e.g., macrophagescontaining Mycobacterium tuberculosis, Mycobacterium leper (leprosy), orSalmonella typhi (typhoid fever).

We turn now to the case of the second embodiment herein where thepathologically proliferating cells are pathologic helminths. The term“pathologic helminths” is used herein to refer to pathologic nematodes,pathologic trematodes and pathologic cestodes. More detail on theapplicable helminths is set forth at column 12 of U.S. Pat. No.6,057,367 which is incorporated herein by reference.

We turn now to the case of the second embodiment where thepathologically proliferating cells are pathologically proliferatingmammalian cells.

The term “pathologically proliferating mammalian cells” as used hereinmeans cells of the mammal that grow in size or number in said mammal soas to cause a deleterious effect in the mammal or its organs. The termincludes, for example, pathologically proliferating cancer cells, thepathologically proliferating or enlarging cells causing restenosis, thepathologically proliferating or enlarging cells causing benign prostatichypertrophy, the pathologically proliferating cells causing myocardialhypertrophy and proliferating cells at inflammatory sites such assynovial cells in arthritis. Pathologically proliferating cancer cellsinclude the cell proliferation in Hodgkin's disease, in small cell lungcancer, in cancer of the breast, and in testicular and prostate cancer.

The inhibitors of glutathione-dependent formaldehyde dehydrogenase forthis second embodiment herein are those described above.

The therapeutically effective amount for this second embodiment means aglutathione-dependent formaldehyde dehydrogenase inhibiting amount invivo which is an antiproliferative effective amount. Suchantiproliferative effective amount as used herein means an amountcausing reduction in rate of proliferation of at lest 10%.

In general, the dosage, i.e., the therapeutically effective orantiproliferative effective amount, ranges from 1 μg to 10 g/kg andoften ranges from 10 μg to 1 g/kg or 10 μg to 100 mg/kg body weight ofthe patient being treated, per day.

The patients are mammals including humans.

The preferred route of administration in respect to inhibiting growth ofmicrobes is oral administration although other routes of administrationincluding parenteral and topical are useful. Topical administration isespecially useful for exposed infections, e.g., fungal infections suchas athlete's foot, viral infections such as herpes and microbe-causedoral or skin lesions. For inhibiting the growth of helminths, thepreferred route of administration is oral administration although otherroutes of administration including parenteral are useful. For inhibitingthe growth of pathologically proliferating cancer cells, the route ofadministration can be oral or parenteral and local administration ispossible, for example, by infusion directly into a tumor or into theblood vessels delivering blood to the tumor, or by forming the agentinto a slow release pellet or into a polymer matrix and then implantingthe pellet or polymer matrix device in or on the tumor. The preferredroutes of administration in the case of inhibiting growth ofpathologically proliferating mammalian cells that would cause restenosisis from attachment on a stent implanted in angioplasty but systemicincluding oral and intravenous administration can be acceptable. Thepreferred route of administration in the case of inhibiting growth ofpathologically proliferating mammalian cells causing benign prostatichypertrophy is from attachment on a prostatic implant or by localinjection.

Where the pathologically proliferating cells comprise pathologicbacteria or fungus and the patient is afflicted with a bacterial orfungal infection, the administering kills or reduces the growth of thepathologic bacteria or fungus. Where the pathologically proliferatingcells are pathologically proliferating mammalian cells, theadministering kills or reduces the growth of the pathologicallyproliferating mammalian cells.

Preferred treating agents for the second embodiment includeD-glutathione, ribavirin, D-glutathione together with ribavirin, andmycophenolic acid. D-Glutathione can be given, for example, inhaled at1-10 mM concentration for viral pneumonia; orally at a dose of 0.2 to 2grams daily for pinworm and Hodgkin's disease, to inhibit restenosisafter angioplasty and to resolve benign prostatic hypertrophy; andintravenously at 100 to 300 mg/kg for squamous cell lung cancer. Thecombination of D-glutathione and ribavirin is preferably given forbacterial pneumonia (e.g., 100 mg-1 g D-glutathione orally and 1-10 gribavirin inhaled) and for metastatic breast cancer, metastatictesticular cancer and metastatic prostate cancer (e.g., 100-300 grams/kgD-glutathione intravenously up to four times or more a day and 1-10 gribavirin intravenously).

Treatment is continued as long as symptoms and/or pathology ameliorate.

The inhibitor of glutathione-dependent formaldehyde dehydrogenase can beadministered alone for the second embodiment or in combination withconventional therapy for the disorder being treated or in combinationwith newly discovered agents for use for therapy of the disorder treatedand/or in combination with any other agent that imposes nitrosative oroxidative stress.

Agents for selectively imposing nitrosative stress to inhibitproliferation of pathologically proliferating cells in combinationtherapy with GS-FDH inhibitors herein and dosages and routes ofadministration therefor include those disclosed in U.S. Pat. No.6,057,367, the whole of which is incorporated herein.

Supplemental agents for imposing oxidative stress (i.e., agents thatincrease GSSG (oxidized glutathione) over GSH (glutathione) ratio orNAD(P) over NAD(P)H ratio or increase thiobarbituric acid derivatives)in combination therapy with GS-FDH inhibitors herein include, forexample, L-buthionine-S-sulfoximine (BSO), glutathione reductaseinhibitors (e.g., BCNU), inhibitors or uncouplers of mitochondrialrespiration and drugs that increase reactive oxygen species (ROS), e.g.,adriamycin, in standard dosages with standard routes of administration.For example, BSO can be given intravenously or orally at 10-30 grams perday.

We turn now to the embodiment directed to a method of treating a patientin need of increased nitric oxide bioactivity, said method comprisingadministering to said patient a therapeutically effective amount of aninhibitor of glutathione-dependent formaldehyde dehydrogenase. Thisembodiment may be referred to as the third embodiment herein.

In one subset, i.e., the first subset, of the third embodiment, thepatient has a disorder associated with a deficiency in nitric oxide. Theterm “disorder associated with a deficiency in nitric oxide” is usedherein mean disorder where NO deficiency is a feature and the deficiencyconstitutes less NO than the norm or less than the normal NO bioactiveresponse. Disorders associated with a deficiency in nitric oxide includeatherosclerosis, restenosis, and disorders involving deficiency in NO intissues where NO is necessary to keep the tissues alive, e.g.,deficiency in NO in endothelial cells, hepatocytes and certain lungsites, including liver diseases comprising inflammatory liver disorders,including, for example, chronic viral hepatitis B, chronic hepatitis C,alcoholic liver injury, drug (including acetaminophen)-induced liverinjury, primary biliary cirrhosis, autoimmune hepatitis, nonalcoholicsteatohepatitis and liver transplant rejection. For atherosclerosis, theNO deficiency can be detected in serum or in blood vessel responses. Forrestenosis, the NO deficiency is inherent in the injurious event whichremoves the source of NO. For liver disorders, the NO deficiency isdetected in liver tissue.

The inhibitors of glutathione-dependent formaldehyde dehydrogenase forthe first subset of the third embodiment are those described above.

The term “therapeutically effective amount” for the first subset of thethird embodiment means a glutathione-dependent formaldehydedehydrogenase inhibiting amount in vivo that causes amelioration of thedisorder being treated or protects against risk associated with thedisorder. For example, for treating atherosclerosis, a therapeuticallyeffective amount is a blood vessel dilating effective amount. Fortreating restenosis, a therapeutically effective amount is a restenosisinhibiting effective amount. For treating liver diseases and disorders,an effective amount is a liver tissue inflammation ameliorating amount.

In general, the dosage for the first subset of the third embodiment,i.e., the therapeutically effective amount, ranges from 1 μg to 10 g/kgand often ranges from 10 μg to 1 g/kg or 10 μg to 100 mg/kg body weightof the patient, per day.

The patients include mammals including humans.

The preferred routes of administration are oral and parenteral includingintravenous with coating on an implanted stent being very appropriatefor treating restenosis.

A preferred agent for treating a patient with atherosclerosis isD-glutathione administered by oral route at a dosage ranging from 0.5 to100 mg/kg.

A preferred agent for treating a patient having or at risk forrestenosis is D-glutathione administered on a stent at a dosage rangingfrom 1 nanomole to 100 micromoles.

A preferred agent for treating liver diseases is D-glutathioneadministered by intravenous route of administration at a dosage rangingfrom 1 to 1,000 mg/kg.

Treatment is continued for the first subset of the third embodiment aslong as symptoms and/or pathology ameliorate.

In a second subset, i.e., the second subset of the third embodiment, thepatient has a disorder where NO bioactivity is beneficially increasedfor pharmacological effect. For example, in hypertension, NO productionmay or may not be normal but in either case, pharmacological amounts arerequired to reverse the blood pressure increase, or in restenosis theblood vessel may heal with restoration of NO production, but more NO maybe required to treat process (so no more smooth muscle proliferation).Another disorder that is embraced in the second subset of the thirdembodiment is heart failure. Still another disorder that is embraced inthe second subset of the third embodiment is angina.

The inhibitors of glutathione-dependent formaldehyde dehydrogenase forthe second subset of the third embodiment are those described above.

The term “therapeutically effective amount” for the second subset of thethird embodiment means a glutathione-dependent formaldehydedehydrogenase inhibiting amount in vivo that causes amelioration of thedisorder being treated or protects against risk associated with thedisorder. For example, for treating hypertension, a therapeuticallyeffective amount is a blood pressure lowering effective amount. Forpreventing occurrence of smooth muscle proliferation after a bloodvessel is healed, a therapeutically effective amount is a smooth muscleproliferation inhibiting amount. For treating heart failure, atherapeutically effective amount is an apoptosis inhibiting effectiveamount. For treating angina, a therapeutically effective amount is anangina ameliorating effective amount.

In general, the dosage for the second subset of the third embodiment,that is, the therapeutically effective amount, ranges from 1 μg to 10g/kg and often ranges from 10 μg to 1 g/kg or 10 μg to 100 mg/kg bodyweight of the patient, per day.

The patients include mammals including human.

The preferred routes of administration are oral and parenteral includingintravenous with coating on an implanted stent being appropriate toinhibit smooth muscle proliferation after a blood vessel is healed inrespect to restenosis.

The preferred agents, dosages and routes of administration in connectionwith treating hypertension and inhibiting smooth muscle cellproliferation are those described in conjunction with treatinghypertension and restenosis vis-a-vis other embodiments herein asdescribed above. Preferred agent for treating heart failure or angina isD-glutathione administered orally at 0.5 to 100 mg/kg.

Treatment is continued for the second subset of the third embodiment foras long as symptoms and/or pathology ameliorate.

The inhibitor of glutathione-dependent formaldehyde dehydrogenase can beadministered alone for the third embodiment or in combination withconventional therapy for the disorder being treated or in combinationwith newly discovered agents for use for therapy of the disorder treatedand/or in combination with any other agent that imposes nitrosativestress or which is functional for pharmacological delivery of NO or NOrelated components for therapeutic application or which upregulatesendogenous NO.

The agents for posing nitrosative stress for this case of the thirdembodiment and dosages and routes of administration therefor are thosedescribed for the second embodiment compatible with treatment of thedisorders of the third embodiment.

The agents functional for pharmacological delivery of NO or NO relatedcompound have the moiety —RNO_(x) where R is N, C, S, O or transitionmetal and x is 1 or 2. These include known agents, e.g., nitroglycerinor nitroprusside used in conventional amounts with conventional routesof administration.

The agents for upregulating endogenous NO include cytokines, e.g., tumornecrosis factor alpha, interferon gamma and interleukin 1β used inconventional amounts with conventional routes of administration, toactivate or upregulate NO synthase to make NO, and substrates for NOsynthase, e.g., L-arginine (e.g., in an amount of 2-10 grams, e.g., 6grams per day administered systemically).

For example, patients with angina or hypertension treated with nitrates,i.e., nitroglycerin or nitroprusside standard of care respectively, whoare not responding adequately can be given GS-FDH inhibitor, e.g., 1 mMD-glutathione, to increase NO levels.

For example, patients with angina can be treated with nitroglycerinadministered in conventional amounts with conventional route ofadministration concomitantly with inhibitor of glutathione-dependentformaldehyde dehydrogenase, e.g., D-glutathione administered orally at0.5 to 100 mg/kg or any of the other GSD-FDH inhibitors specificallyrecited above.

Moreover, for example, patients with malignant hypertension from heartfailure treated with nitroprusside in conventional amounts withconventional route of administration can be concomitantly administeredGS-FDH inhibitor, e.g., D-glutathione administered orally at 0.5 to 100mg/kg or any of the other GS-FDH inhibitors specifically recited above.

The mechanism of the invention is shown by the following backgroundexample and the invention is illustrated by the following workingexamples.

Background Example 1 Detection of S-Nitrosoglutathione ReductaseActivity in E. Coli Lysates

A crude extract (500 μg/ml; E. coli strain RK4936) was incubatedanaerobically with 0.2 mM NADH in the absence or presence of 0.15 mMS-nitrosoglutathione (GSNO), and NADH consumption (absorbance at 340 in)was followed over time. The experiment was performed anaerobically toeliminate non-specific NADH oxidation by diaphorases present in E. colilysates. In addition, K_(M) values, K_(cat) values and K_(cat)/K_(M)values were obtained based on assays using 12 nM enzyme, 0.2 mM NADH and10-500 μM GSNO using as buffers (100 mM; 0.1 mM DTPA), sodium acetate(OH 4-5), MES (pH 6), BisTrisPropane (pH 7) and Tris (pH 7.5-9). Theexperiment showed a GSNO-consuming activity in E. coli lysates that wasdependent on NADH.

Identification of Activity as Glutathione-Dependent FormaldehydeDehydrogenase (GS-FDH)

The GSNO metabolizing activity from E. coli strain RK4936 was purifiedfrom 8 liters of stationary phase cells. A 100,000 g supernatant in 20mM BisTrisPropane (pH 7) was applied to a 5×40 cm Q-Sepharose column andeluted with a linear NaCl gradient in 20 mM BisTrisPropane (pH 7).Active fractions were pooled, adjusted to 1 M (NH₄)₂SO₄, and applied toa 2.5×20 cm column of Butyl Sepharose. Elution was done with adecreasing (NH₄)₂SO₄ gradient from 1 to 0 M. After gel filtration on aHiPrep 16/10 desalting column (Pharmacia), the enzyme was fartherpurified on a MonoQ column. Active fractions were applied to a 1.6×10 cmcolumn of AMP Sepharose, washed with 0.15 M NaCl, and eluted with 20 mMNAD⁺. The protein was pure as judged by SDS-PAGE (yield: 0.52 mg).Limited N-terminal sequencing after blotting onto a PVDF membraneidentified the protein as the glutathione-dependent formaldehydedehydrogenase.

Detection of GS-FDH in Mouse Macrophages

Mouse macrophages RAW264.7 cells (cell line obtained from the ATCC) werestimulated with interferon-gamma and lipopolysaccharide (LPS) for 18hours as described in Liu, L., et al. Proc. Natl. Acad. Sci. USA 96,6643 (1999). S-Nitrosothiol levels in the whole lysate, in the fractionof the lysate that passed through a Bio-Gel P-6 column and in thefraction that passed through a 5 kDa out-off ultrafiltration membranewere measured by photolysis-chemiluminescence as described in Mannick,J. G., et al., Science 284, 651 (1999). Amounts of S-nitrosothiol (pmol)were normalized against protein content (mg) of the whole lysate. Dataobtained were the mean±SE of three independent experiments. It was foundthat S-nitrosothiols larger than 5 kDa (high-mass) were present in highamounts in the cells lysate, whereas low mass S-nitrosothiols (<5 kDa)were not detectable (limit of sensitivity 1 pmol S-nitrosoglutathione(GSNO)).

Since GSNO (˜300 pm/10⁶ cells) formed in the extracellular medium ofinterferon-gamma/LBS-treated RAW 264.7 cells that were incubated withglutathione as described in Akaike, T., et al., J. Biochem (Tokyo) 122,459 (1997), and glutathione is the predominant source of intracellarthiol (Kosower, N. S., et al., Int. Rev. Cytol. 54, 109 (1978)), weconcluded that the reason for the lack of GSNO in our experiment wasthat it was either rapidly exported or metabolized.

Rates of nitrosothiol accumulation in the medium of RAW 264.7 cells wasfound to be very slow at the limits of detection, and this result wasconsistent with what was found in Kosower, N. S., et al., Int. Rev.Cytol. 54, 109 (1978).

To test whether GSNO was being rapidly metabolized, GSNO was incubatedin reaction buffer supplemented with NADH or in cell lysates with andwithout NAD. Cells were homogenized by sonication in a solutioncontaining 20 mM Tris-HCl (pH 8.0), 0.5 mM EDTA, 0.1% NP-40 and 1 mMphenylmethylsulfonyl (PMSF). To detect GSNO-metabolizing activity, 0.86mg/ml RAW 264.7 lysate was incubated with 100 μM GSNO in reaction buffer(20 mM Tris-HCl (pH 8.0), 0.5 mM EDTA) with 0 or 200 μM NADH at roomtemperature for various times. Nitrosothiol levels in the reactionmixture were measured by the Saville assay as described in Stamler, J.S., Science 276, 2034 (1997). GSNO-dependent NADH consumption was alsomeasured using either absorbance (OD 340 nm) or fluorescence (340/455nM) measurements of NADH. The results showed that GSNO was quicklymetabolized when it was incubated with extracts from restingmacrophages. The same result of GSNO being quickly metabolized wasobtained from cytokine activated macrophages. As in E. coli, the GSNOmetabolizing activity required NADH and was ineffective at metabolizingalternate nitrosothiols.

GSNO-metabolizing activity of RAW 264.7 cells was recovered in a singlefunction following anion exchange on a MonoQ column. Furtherpurification over a 5′ AMP Sepharose column yielded a single band ofabout 43 kDa in a silver-stained SDS-PAGE gel. To purify the GSNOreductase, the cell lysate was first subjected to stepwise ammoniumsulfate precipitation. The GSNO) reductase activity was precipitatedbetween 45-75% ammonium sulfate. After dialysis against 20 mM Tris-HCl(pH 8.0), the proteins were fractionated on a MonoQ column withincreasing concentrations of NaCl. The GSNO reductase activity wasfinally purified with a 5′ AMP Sepharose 4B affinity column (AmershemPharmacia) in 20 mM phosphate buffer. SDS-PAGE and Coomassie bluestaining gave the single band of about 43 kDa.

The purified protein was digested by trypsin and the resulting peptideswere analyzed by mass spectrometry. Peptide mass database search withGPMAW program identified the protein as mouse GS-FDH(SP-P28474) withhigh scores, matching 8 fragments (42% coverage) to mouse GS-FDH.

The purified protein also oxidized the formaldehyde-glutathione adduct,S-hydroxymethylglutathione, previously thought to be the major enzymesubstrate. The specific activity for this oxidation was only about 6% ofthe GSNO reducing activity. Thus, GSNO is the preferred substrate ofthis enzyme.

Kinetic analysis revealed that the purified protein has a K_(M) of 20 μMand an estimated K_(cat) 5,600 min⁻¹ for GSNO. The stoichiometry of GSNOto NADH was found to be one to one.

In summary, the GSNO reductase activity of mouse macrophage cells isremarkably high and specific toward GSNO, and this activity constitutesa major metabolic pathway for GSNO in mouse macrophages. The dataindicates that by eliminating endogenous GSNO, GS-FDH protects fromnitrosative stress.

Detection in Other Mouse Cells

NADH-dependent GSNO reductase activity was also detected in mouseSVEC4-10 endothelial cells (cell line obtained from the ATCC) and mouseSV40LT-SMC smooth muscle cells (cell lie obtained from the ATCC).

Detection in Human Cells

Testing for NADH-dependent GSNO reductase activity was carried out onhuman HeLa cells, human epithelial A549 cells, and human monocyte THP-1cells. The cells were from cell lines obtained from the ATCC. Cells werehomogenized by sonication in a solution containing 20 mM Tris-HCl (pH8.0), 0.5 mM EDTA, 0.1% NP-40 and 1 mM phenylmethylsulfonyl fluoride(PMSF).

GSNO (200 μM) was incubated with 0 or 4 μg/ml of the cell lysates inreaction buffer (20 mM Tris-HCl (pH 8.0), 0.5 mM EDTA) supplemented withNADPH (300 μM), NADPH (300 μM), glutathione (GSH, 2 mM), and ascorbicacid (ASA, 500 μM) in combinations as set forth in the table below, at37° C. for 5 minutes. After precipitation of the reaction mixtures withtrichloroacetic acid (8.3% final concentration), the supernatants werediluted threefold in the buffer and nitrosothiol levels in the reactionmixture were measured by the Saville assay as described in Stamler, J.S., Science 276, 2034 (1997). Data are the mean of two independentexperiments.

Data are set forth in the table below where GSNO metabolizing activityis shown as percent of that in the starting material and ASA isascorbate and GSH is glutathione (ascorbate and glutathione are redoxco-factors that have been proposed as decomposing GSNO).

TABLE No lysate HeLa A549 THP-1 Buffer only 6.2 29.9 14.7 18.8 +NADH 0.084.2 72.3 90.8 +NADPH + GSH + ASA 0.7 35.4 43.9 22.6

The results indicate GSNO reductase activity was widespread in varioushuman cell lines.

It is Already Known that GS-FDH is Highly Conserved from Bacteria toMammals

Publications indicating that GS-FDH is highly conserved from bacteria tofungi and mammals are (1) Danielsson, O., et al., Proc. Natl. Acad. Sci.USA 91, 4980 (1994); (2) Shafquat, J., et al., Proc. Natl. Acad. Sci.USA 93, 5595 (1996); (3) Wach, A., et al., Yeast 10, 1793 (1994); (4)Gutheil, W. G., et al., Biochemistry 31, 475 (1999); (5) Wehner, E. P.,et al., Mol. Gen. Genet. 237, 351 (1993), and (6) Barber, R. D., et al.,J. Bacterial 178, 1386 (1996). The proteins from E. coli, S. cerevisiaeand mouse macrophages share over 60% of amino and sequence identity.

Mutant Cells Deficient on GS-FDH Accumulate Increased Levels of GSNO andProtein Nitrosothiols

In cases of conservation of genetic function, because of ease of geneticmanipulation, yeasts are often used to identify and characterizemammalian genes. See Cardenas, M. E., et al., Clin. Microbiol. Rev. 12,583 (1999).

As a first step in using this thesis, yeast strain Y190 cells (Clontech;Genbank Accession No. Z74216) were tested for GSNO reductase activity.GSNO was incubated in buffer or lysate of the yeast cells. The testshows the yeast strain Y190 cells have robust GSNO reductase activity.

The entire open reading frame of the GS-FDH/GSNO reductase genedescribed in Wehner, E. P., et al., Mol. Gen. Genet. 237, 351 (1993)(SFA1/YDL168w) was replaced with dominant selectable cassettes Kan 2(resistant to G418) or hphMX (resistant to hydromycin) by directtargeting as described in Wach, A., et al., Yeast 10, 1793 (1994).KanMX2 is described in Wach, A. et al., Yeast 10, 1793 (1994). hphMX isdescribed in Goldstein and MeCuskler, Yeast 15, 1541 (1991).Comprehensive analyses of sfa1(gs-fdh) mutant yeast (4G418^(r) and4hygromycin^(r) clones) showed they lost all GSNO reductase activity.

Replacement of the SFA1 gene with KanMX2 and hph was also carried out inthe diploid yeast strain JK93d. After the cassette positively targetedone of the two alleles, diploid cells resistant to either G418 orhydromycin were induced to sporulate. Haploid clones of wild-type andmutant cells obtained by tetrad dissection revealed the loss of GSNOreductase activity co-segregated with resistance to antibiotics(G418^(r) or hyg^(r)). A 2:2 ratio was seen in all four complete tetradsstudied.

When wild-type Y190 yeast cells were treated with GSNO, the yeastaccumulated only low levels of nitrosothiols, all of which were highmass (i.e., retained by a 5 kDa cut-off filter). In contrast, thenitrosothiol levels were more than 11-fold higher in isogenic sfa1mutants (both sfa1Δ::G418^(r) and sfa1Δ::hygromycin^(r)) and substantialamounts of low-mass nitrosothiols were detected. This low-massnitrosothiol pool was completely metabolized by addition of GSNOreductase, thus identifying the low-mass nitrosothiol as GSNO.

Growth of sfa1 mutant cells (both G418^(r) and hygromicin^(r) clones)was markedly inhibited by GSNO at concentrations that had little effecton wild-type Y190 cells.

The mutant cells were no more sensitive to the toxic effects of H₂O₂than wild-type Y190 cells.

These data show that GS-FDH/GSNO reductase is essential for protectionof nitrosative stress in yeast; whereas it offers no resistance tooxidative stress. The protection is conferred by metabolizing GSNOdirectly.

NO Synthase Activation and GSNO Measurement in Mouse Hepatoytes

Hepatocytes of wild-type mice and their GS-FDH^(−/−) littermates wereharvested after perfusion of the livers with Liberase (Roche), andpurified by repeated differential sedimentation at 50×g, followed bycentrifugation over a 30% Percoll solution. Hepatocytes (over 95%viability by trypan blue exclusion) were plated at a density of 2×10⁴cells/cm² in gelatin-coated flasks, and incubated for 24 hours. Thecells were then cultured in the presence of recombinant mouse tumornecrosis factor-α (500 units/ml), interferon-gamma (100 units/ml),interleuken-1β (200 units/ml) and lipopolysaccharide (LPS, 10 μg/ml; E.coli 0128:B12; Sigma) for 14 and 40 hours. Nitrosylation levels inhepatocytes were measured as described in Eu, J., et al., Biochemistry39, 1040-1047 (2000). Data showed total nitrosothiol but not alternativeNO complexes, were approximately 50% higher in cells deficient in GS-FDHand that the majority of nitrosothiol was bound to protein. Moreover,whereas GSNO was barely detectable in wild-type hepatocytes, the levelsincreased by 60-175% in GS-FDH⁴ cells. It is known that such increasesprotect from liver injury. See Liu, L. and Stamler, J. S., Cell Deathand Differentiation 6, 937-942 (1999). The data suggest that inhibitingGS-FDH will protect liver cells from ischemic injury.

Detection in Tissues

Liver tissues from wild-type and GS-FDH-deficient mice were homogenizedin an enzyme reaction buffer (phosphate-buffered saline, GIBCO14190-144, supplemented with 0.1% NP-40 and 1 mM PMSF). GSNO (200 μM)was incubated with 0 or 3 μg/μl of the liver homogenate in buffersupplemented with NADH (300 μM), buffer supplemented with NADPH (300μM), buffer supplemented with glutathione (2 mM), buffer supplementedwith ascorbic acid (500 μM) or buffer supplemented with NADPH (300 μM),glutathione (2 mM), and ascorbic acid (500 μM), at 37° C. for 5 minutes.After precipitation of the reaction mixture with trichloroacetic acid(8.3% final concentration), the supernatants were diluted three-fold inthe buffer and nitrosothiol levels were measured by the Saville assay asdescribed in Stamler, J. S., Science 276, 2034 (1997). Data are the meanof two independent experiments. These studies showed that theNADH-dependent GSNO reductase activity dominates alternativedecomposition reactions that operate in simplified in vitro systems.

GS-FDH Deficiency Protects in the Case of Liver Injury

Cells dislodged by perfusion and purified as described above, from aninjured mouse liver (injured by cytokine administration), treated with 1millimolar N^(G)-methyl-L-arginine nitric oxide synthase inhibitor (20%activity left), were found to die.

Cells that were the same but deficient in GS-FDH died to a lesserextent, indicating that inhibiting GS-FDH protects injured liver cells.

Working examples illustrating the invention, follow.

Example I

A 25-year-old white female presents to the Emergency Room wheezing withan FEV1 of 1 liter. She is given an intravenous infusion ofD-glutathione 50 mg/kg Q.6 hours and her breathing improves. She issubsequently given inhaled D-glutathione (nebulized solution of 10 mMD-glutathione in 3 cc of normal saline) to be taken twice daily.

Example II

A 17-year-old male with cystic fibrosis presents to the Emergency Roomwith difficulty breathing and a fever. He is given an inhaled treatmentof S-nitrosoglutathione (10 mM in 3 cc normal saline) in combinationwith D-glutathione (3 mM in 10 cc normal saline). His symptoms resolveover the following day.

Example III

A 40-year-old female develops ARDS as a complication of urosepsis. Sheis intubated and transferred to the Intensive Care Unit. Her blood gasshows a PO, of 60 mm Hg on 100% oxygen. She is given an inhaled dose of10 mM D-glutathione (in 3 cc normal saline) with an increase in PO₂ to80 mm/Hg, seen over 1 hour.

Example IV

A 70-year-old male status post CABG×2 presents with unstable angina. Heis treated with betablockers and nitrates but continues to experiencechest pain. An intravenous infusion of 100 mg per kg D-glutathione isgiven Q6 hours with relief of his symptoms. This k an example oftreating heart disease.

Example V

A 60-year-old white male presents to his primary care physician. Onroutine physical exam his blood pressure is 160/90. The patient is begunon 600 milligrams of D-glutathione BID. On follow-up examination threeweeks later, his blood pressure is 140/80.

Example VI

The heart disease treated in Example IV is ischemic coronary syndrome,so Example IV is also an example of treating ischemic coronary syndrome.

Example VII

A 60-year-old white male who undergoes cardiac catheterization and anacetylcholine infusion, shows impaired relaxation (earliest marker ofatherosclerosis). The patient is treated with D-glutathione, 600 mg TIDfor a week. Upon retesting, the patient shows an improved response toacetylcholine.

Example VIII

A 65-year-old male with intractable angina is administered ribavirn as 6grams diluted to a final volume of 300 cc in sterile water and injectedat a final concentration of 20 milligrams per milliliter, given dailyfor seven days. The patient's angina had improved by six weeks. This isan example of treating disease characterized by angiogenesis.

Example IX

A 43-year-old female presents with shortness of breath and hemoptysis. AVQ scan shows a pulmonary embolism and the patient is begun on inhaledribavirn, 6 grams diluted in 300 cc and administered by a small particleaerosol generator. Treatment is carried out for 12 hours/day for threedays. The patient shows improvement in symptoms. This is an example oftreating a disorder where there is risk of thrombosis occurring.

Example X

A 72-year-old white male presents with chest pain seven dayspost-angioplasty and cardiac catheterization reveals restenosis. A Nirstent coated with mycophenolic acid (drug concentration of 30% perpolymer) was deployed with successful results. The patient wasdischarged the following day, and did well.

Example XI

A 17-year-old male with chronic psoriasis presents at the dermatologist.A paste comprised of acetylsalicylic acid, 2%, combined with 5%ribavirin was applied topically for 8 hours a day. This was repeateddaily for two weeks at which time the psoriatic lesion had resolved.This is an example of treating a patient with a chronic inflammatorydisease.

Example XII

A 15-year-old boy presents to the Emergency Room having ingested a toxicdose of Tylenol. Liver fixation tests are elevated. The doctor infusesD-glutathione at 200 milligrams per kilogram every 6 hours with gradualnormalization of liver function over the following week. This is anexample of treating a patient for a disease where there is risk ofapoptosis occurring.

Example XIII

A 70-year-old diabetic with a history of impotence presents to hisurologist. He is treated with Viagra but shows no improvement. Hisphysician adds topical mycophenolic acid (5% paste) with good results.

Example XIV

A 40-year-old alcoholic male presents with swollen abdomen and elevatedLSTs. The patient is infused with D-glutathione at 200 milligrams perkilogram every 6 hours for two weeks. The swelling and elevated LSTs arereduced.

Example XV

A 52-year-old female presents with pneumonia to the Emergency Room. Asputum culture grows out pseudomonas resistant to antibiotics and she isgiven D-glutathione, 600 milligrams TID, and inhaled ribavirin 6 gmdaily for ten days with a resolution of pneumonia.

Example XVI

A 9-year-old presents to her pediatrician who diagnosis ringworm Sheapplies topical ointment comprised of 5% ribavirin acid daily for a weekand the skin lesion resolve.

Example XVII

A 52-year-old female status post lung transplant is admitted to thehospital with viral pneumonia. She is given inhaled 1)-glutathione, 3mM/3 cc normal saline four times a day with resolution of symptoms ofcough and shortness of breath.

Example XVIII

A tape test done by a pediatrician conforms the suspected diagnosis ofpinworm The child is given 1 gram of D-glutathione daily for three daysand the signs and symptom resolve.

Example XIX

A routine chest X-ray shows the presence of a right upper lobe lung massin a 55-year-old smoker. Bronchoscopy shows a mass at the take off tothe right upper lobe and a biopsy confirms diagnosis of squamous cellcancer. A further work-up shows the patient to be stage 3B. The patientis treated with a regimen including three weeks of D-glutathioneintravenously 200 milligrams per kilogram four times a day for twoweeks. On restaging, the patient is 3A and thus a candidate for surgery.

Example XX

A 25-year-old has a biopsy done on an axillary node which establishesthe diagnosis of Hodgkins disease. The patient is given 1 gram ofD-glutathione three times a day for a month with resolution ofadenopathy.

Example XXI

A 55-year-old female with metastatic breast cancer, unresponsive toconventional measures, is begun on combination of intravenous ribavirin6 grams a day for seven days and intravenous D-glutathione, 200 gramsper kilogram QID. Symptoms of bone pain in her back resolve after twodays.

Example XXII

A 30-year-old white male presents with a testicular mass and metastasisto lung and brain. The diagnosis is metastatic testicular cancer. Thepatient unresponsive to conventional measures, is begun on combinationof intravenous ribavirin 6 grams a day for seven days and intravenousD-glutathione, 200 grams per kilogram QID. Symptom of headache resolveafter two days.

Example XXIII

A 65-year-old male presents with highly elevated PSA and metastasis tobone. The diagnosis is metastatic prostate cancer. The prostate canceris treated with conventional therapy. However, the metastasis to bone isunresponsive to conventional measures. The patient is begun oncombination of intravenous ribavirin 6 grams a day for seven days andintravenous D-glutathione, 200 grams per kilogram QID. Symptoms of bonepain in back resolve after two days.

Example XXIV

A 65-year-old male has a stent placed for 90% lesion of the leftanterior descending coronary artery. He is sent home on a regimenincluding D-glutathione, 600 mg P.O. three times a day for a month anddoes well.

Example XXV

A 55-year-old male complaining of urinary frequency and hesitancy(diagnosis: benign prostatic hypertrophy) is begun on D-glutathione, 600mg three times a day. His symptoms gradually resolve over the followingthree months.

Example XXVI

A 27-year-old female with acute viral myocarditis develops severe heartfailure. Her EF is 10%, and she is unresponsive to conventional therapy.She is placed on the transplant list. Infusion of D-glutathione, 1 gramTED for 10 days, stabilized her deteriorating course.

Example XXVII

A 60-year-old male presenting with angina is treated with nitroglycerine(0.6 mg sublingually) and responds only partly as evidenced bypersistent chest pain. The patient is concomitantly started onD-glutathione, 600 mg TIE, with total relief of symptoms. The otherGS-FDH inhibitors specifically recited above can be substituted forD-glutathione in appropriate amounts with similar results.

Example XXVIII

A 65-year-old male presents with heart failure and malignanthypertension. Blood pressure is reduced from 240 to 200 systolic withnitroprusside (1.0 μg/kg/min IV infusion, for 10 minutes) and is reducedfurther to 160 by coadministration of GS-FDH inhibitor, e.g.,D-glutathione, 600 mg, TID.

VARIATIONS

Many variations on the above will be obvious to those skilled in theart. Thus, the scope of the invention is defined by the claims.

1. A method for treating a patient afflicted with a disorder amelioratedby NO donor therapy, said therapy comprising administering to saidpatient a therapeutically effective amount of an inhibitor ofglutathione-dependent formaldehyde dehydrogenase wherein the inhibitoris D-glutathione or mycophenolic acid.
 2. The method of claim 1 wherethe patient is afflicted with a breathing disorder.
 3. The method ofclaim 2 where the breathing disorder is asthma.
 4. A method for treatinga patient in need of increased nitric oxide bioactivity, said methodcomprising administrating to said patient a therapeutically effectiveamount of an inhibitor of glutathione-dependent formaldehydedehydrogenase.
 5. The method of claim 4 where the patient is afflictedwith atherosclerosis.
 6. The method of claim 4 where the patient isafflicted with restenosis.
 7. The method of claim 4 where the patient isafflicted with an inflammatory liver disease.
 8. The method of claim 4where the patient is afflicted with heart failure.